Streptococcus pneumoniae is an important pathogen, causing invasive diseases such as pneumonia, meningitis, and bacteraemia. Even in regions where effective antibiotic therapy is freely available, the mortality rate from pneumococcal pneumonia can be as high as 19% in hospitalized patients. In developing countries, in excess of three million children under the age of five years die each year from pneumonia, of which S. pneumoniae is the most commonly identified causative agent. S. pneumoniae also causes less serious, but highly prevalent infections such as otitis media and sinusitis, which have a significant impact on health-care costs in developed countries. Otitis media is especially important in young children, while sinusitis affects both children and adults.
Currently, licensed anti-pneumococcal vaccines are based on formulations of various capsular polysaccharide antigens derived from highly prevalent strains. Serotypes that most commonly cause invasive pneumococcal infection appear to differ somewhat in various regions of the world. In North America in the pre-vaccination era, serotypes 4, 6B, 9V, 14, 18C, 19F and 23F were the seven most common serotypes causing invasive disease in children aged ≦5 years of age (Butler, et al. J. Infect. Dis. 171 (4): 855-889 (1995)). These serotypes were reported to be responsible for 70-88% of invasive disease in these children and accounted for 100% of S. pneumoniae with high-level penicillin-resistance.
Two types of pneumococcal vaccines are in clinical use: the 23-valent Pneumococcal Polysaccharide Vaccine (23-PPV) and the 7-valent Pneumococcal Conjugate Vaccine (PCV7) (Siber, et al. Pneumococcal Vaccines: The Impact of Conjugate Vaccine. Washington D.C.: ASM Press; 2008). The polysaccharide antigens in 23-PPV elicit a T-cell-independent immune response, resulting in poor immunologic memory. Additionally, while 23-PPV confers 60-80% protection against invasive pneumococcal disease (IPD) in adults and the elderly, immunity wanes substantially after 5 years and it is poorly immunogenic in children ≦2 years of age. Robust T-cell responses with immunologic memory is observed in young children vaccinated with PCV7 (Prevnar®, Wyeth Pharmaceuticals, Inc.) Prevnar 13 (PCV13), which includes the serotypes of PCV7 and serotypes 1, 3, 5, 6A, 7F, and 19A was recently recommended for approval by a Food and Drug Administration advisory committee on vaccines and related biological products. Studies are also underway on a ten-valent vaccine (Synflorix, GSK) containing polysaccharides of ten pneumococcal serotypes (1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F) conjugated to a carrier protein.
Despite their resounding success and significant public health impact, anti-pneumococcal conjugate vaccines have also had some well known shortcomings, including the complexity of conjugate vaccine production, which increases manufacturing costs. More important, however, is the finding that polysaccharide-based conjugate vaccines protect only against infections caused by bacteria that express the particular capsule-type represented by the vaccine. This is a problem in regions such as Latin America, Asia and Africa, in which the serotypes represented by PCV7 are responsible for much less invasive disease than elsewhere in the world (Black, et al. In: Plotkin S A, et al. eds. Vaccines, 5th Edition. WB Saunders. Chapter 23 (2008); Garcia, et al. Rev. Panam. Salud. Publica. 19(5):340-8 (2006); Lagos, R. Pediatr. Infect. Dis. J. 21 (12): 1115-23 (2002)). This need may be addressed by the new generation of conjugate vaccines, the 10- and 13-valent PCV vaccines (PCV-10 and PCV-13), currently in development and/or licensure stage.
Regional issues (e.g., limited serotype coverage, the potential for replacement disease with non-vaccine serotypes, capsular switching, carrier-induced suppression, and manufacturing and supply constraints are understood by those of skill in the art to present significant problems in vaccinating worldwide populations. It is known that S. pneumoniae is antigenically and clonally diverse (Hanage, et al. Infect. Immun. 73(1):431-5 (2005)), with a single pneumococcal serotype typically including a number of genetically divergent clones. Pneumococcal proteins are known to be much more conserved between serotypes and have thus been considered as potential vaccines.
Pneumolysin has been reported to be an intracellular protein that causes a variety of toxic effects in vivo when released upon lysis of pneumococci. The protein is highly conserved in both amino acid sequence and antigenicity among clinical isolates, thus satisfying some basic criteria for its use as a vaccine antigen (Paton, et al. Infect. Immun. 40(2) 548-52 (1983); Lock, et al. Microb. Pathog. 5(6): 461-67 (1988)). However, it has inherent hemolytic properties, and mutants have therefore been developed and studied for their potential as vaccines. Historically the most commonly studied pneumolysin mutant is PdB, containing a single amino acid change, Trp433Phe (Paton, et al. Infect. Immun. 59(7):2297-304 (1991); Lu, et al. Infect. Immun. 77(5): 2076-83 (2009); Ogunniyi, et al. Infect Immun. 75(1):350-7 (2007); Berry, et al. Infect. Immun. 63(5): 1969-74 (1995); Berry, et al. Infect. Immun. 67(2): 981-85 (1999)).
Other PLY mutants, including ΔAla146 with a deletion of amino acid 146, and ΔAla146R147, have been recently described (Kirkham, et al. Infect. Immun. 74(1): 586-93 (2006)). Both ΔAla146 and ΔAla146R147 were shown to lack haemolytic activity against human erythrocytes. Alum-adjuvanted ΔAla146 was also shown to be as protective as alum-adjuvanted wild-type PLY.
Although there were differences in the mouse strains used, the S. pneumoniae serotypes used and the routes of immunization, compilation of results from these studies using sepsis models have indicated that PdB or ΔAla146 prolong survival of mice when compared with placebo control groups. In the pneumonia model, immunization of mice with PdB yielded a significant decrease in numbers of pneumococci in the lungs of infected mice compared to a placebo control (Briles, et al. J. Infect. Dis. 188(3): 339-48 (2003)). Moreover, in most of these findings, PdB was found to provide a superior protection against a wide variety of strains when used in combination with other virulence factors such as PspA, PspC or PsaA (Lu, supra; Ogunniyi, supra; Berry (1995), supra). Although the PdB mutant provided significant protection in some models, it had the drawback of possessing residual hemolytic activity. However, as mentioned above, ΔAla146 provided both protective immunity and a lack of haemolytic activity.
While several mutant PLY vaccines have been developed, there is a clear need in the art for additional mutants that are both protective and lack haemolytic activity. One such mutant, PlyD1, is described herein. The data presented herein shows that PlyD1 lacks hemolytic activity, generates neutralizing antibodies that inhibit hemolysis by PLY in vitro, and is protective in certain animal models.